The internal consistency of the scale was also assessed. The factor analysis revealed that the scale comprised one factor relevant to shoulder awareness but with fewer items (first 6 items) than the original FreSHAQ. Additionally, the FreSHAQ-GR (both versions) demonstrated statistically significant correlations (Spearman’s r) with participants’ age , the PI-VAS , the SPADI total score and both of its subscales , the QuickDASH , and the PCS . The questionnaire’s internal consistency  was 0.833/0.827 for the 9-item and 0.871/0.867 for the 6-item FreSHAQ-GR. Test–retest reliability was excellent for both versions of the FreSHAQ-GR  = 0.97/0.98 (0.91–0.99/0.94–0.99), and with a low error margin (SEM = 1.31/0.94 and MDC95% = 3.63/2.61). Therefore, the FreSHAQ-GR (6-item version) possesses very good measurement properties and can be administered to Greek-speaking patients with chronic shoulder pain.Shoulder pain is one of the most common musculoskeletal pathologies. The association of shoulder pain with impaired proprioception and bodily self-awareness called for the cross-cultural adaptation of the Fremantle Shoulder Awareness Questionnaire (FreSHAQ) in Greek. The FreSHAQ is a relatively new self-assessment 9-item scale for impaired somatic awareness in patients with shoulder pain. The study included 100 participants (54 women) between 20 and 80 years old, with chronic shoulder pain (>3 months duration), able to comprehend and speak Greek, and recruited from three private physical therapy practices. The cross-cultural adaptation process was followed to develop the Greek version of the questionnaire (FreSHAQ-GR). Further testing for construct validity via exploratory factor analysis and correlational analysis with the Shoulder Pain and Disability Index (SPADI), the Quick Disabilities of Arm, Shoulder, and Hand (QuickDASH), the pain catastrophizing scale (PCS), a pain intensity visual analog scale (PI-VAS) in the last week, and the demographic characteristics followed. Test–retest reliability of the FreSHAQ-GR in a sub-sample of patients (n = 25) was tested upon repeated completion of the scale after a week and examined with the intraclass correlation coefficient (ICC Shoulder pain incidence is ranked high among musculoskeletal-related disorders, with its incidence ranging from 7.7 to 62 per 1000 persons per year (median 37.8 per 1000 persons per year) ,5. ShoulMost shoulder pathologies can be diagnosed at the first visit to primary care when a careful medical history is taken, combined with a proper physical examination ,9,10. ThHowever, chronic shoulder pain develops in a large proportion of patients with acute shoulder pain complaints, irrespective of the cause of initial onset . In chroThe “body self-awareness” construct has been described as complex and multidimensional, and when considered maladaptive in certain states such as chronic pain, it manifests with cognitive distortions of painful body parts and is strongly influenced by mental and emotional processes . Bodily Therefore, the aim of this study was to conduct a cross-cultural adaptation of the FreSHAQ questionnaire in Greek, to determine its degree of reliability upon repeated administration and its structural validity by conducting exploratory factor analysis, and to determine its association with other shoulder pathology-related questionnaires.The study was conducted on 100 adults aged between 20 and 80 years with a diagnosis of painful shoulder pathology lasting for more than three months who, after clinical evaluation, were positive in at least three of the following six clinical tests: (1) Hawkins impingement sign; (2) Neer’s impingement sign; (3) painful arc sign; (4) Jobe’s test; (5) Whipple’s test; (6) shoulder pain elicited by resistance testing during shoulder abduction or external rotation. In addition, participants were excluded based on the following criteria: (1) history of shoulder surgery or planned shoulder surgery; (2) history of fracture, dislocation, or degenerative disease of the shoulder joint; (3) active shoulder range of motion <90 degrees of flexion or abduction or <0 degrees of external rotation; (4) cervical radiculopathy or examination that caused shoulder pain/reduced range of motion after repetitive neck movements; and (5) other serious orthopedic problems . Sp. Sp2,1  . For comThe cross-cultural adaptation process of the FreSHAQ-GR was seamlessly executed, with the scale being rated as clearly phrased and all its items rated as relevant to the patients’ condition and symptomatology. Moreover, in the present study, no “floor” or “ceiling effects” were present for the total scale score, similar to the study by Nishigami et al. , but forRasch analysis was previously conducted for the FreSHAQ-J, revealing the scale’s unidimensionality; however, EFA has not been conducted. In this study, we conducted an EFA of the FreSHAQ-GR with two factors initially extracted: Factor 1 comprising questions 1–6 and 8, and factor 2 containing questions 7 and 9. However, parallel analysis indicated that a single factor should be extracted. Moreover, the internal consistency reliability of McDonald’s ω coefficient of the second factor could not be calculated as it consisted of only two items. Then, item 8 was also removed as it presented low communality. The single factor finally extracted collectively explained 61.44% of the variability in shoulder self-awareness. The FreSHAQ-J was based on the FreBAQ scale, comprising nine items, the first three relating to cognitive and motor neglect, the following two relating to proprioception impairment, and the last four depicting impaired body perception (size and shape) of the painful body part ,21. Howep < 0.001) for both the 9 and 6-item versions of the FreSHAQ-GR with age, the PI-VAS, the SPADI, the QuickDASH, and the PCS. These results confirm previous findings of significant associations between the FreSHAQ-J and pain VAS (r = 0.2), the QuickDASH (r = 0.49), and the PCS (r = 0.38) [To further examine the construct validity of the FreSHAQ questionnaire, the degree of association with patient demographic characteristics but most importantly with other scales examining the clinical status of patients showed highly significant correlations ( = 0.38) . We chos = 0.38) . Since aThe internal consistency of the FreSHAQ-GR measured with two relevant indices  was very good for both the 9- and 6-item versions, with comparable values between the two indices. For the 9-item FreSHAQ-GR, the internal consistency was higher compared to that of the FreSHAQ-J (α = 0.71), with the latter value also acceptable.95%), were at acceptable levels relative to the range of values of the questionnaire. The MDC95% = 3.63 for the 9-item and MDC95% = 2.61 for the 6-item. FreSHAQ-GR in particular represents the smallest amount of true change in the clinical condition of a patient, meaning that scores ±4 for the 9-item and ±3 for the 6-item FreSHAQ-GR of a patient’s initial scores are probably due to a genuine improvement (or deterioration) in their clinical condition.The test–retest reliability of the questionnaire with a time interval between 5 and 7 days in 25 participants was excellent for both the 9 and 6-item versions of the FreSHAQ, with an ICC2,1(95% CI) = 0.97/0.98 (0.91–0.99/0.94–0.99), respectively. In comparison, the test–retest reliability of FreSHAQ-J was also very good, demonstrating an ICC3,1(95% CI) = 0.84 (0.70–0.92) . FurtherSome limitations present in this study were the fact that there were no other cross-cultural adaptations of the FreSHAQ-J, so it was not possible to collect and compare data from other similar studies with the results from the present study. The number of participants utilized satisfied the minimum criteria for EFA analysis. Moreover, the number of participants included in the test–retest reliability study was a quarter of the total sample, which is adequate for this type of investigation . Lastly,As far as future investigations are concerned, there is a need for further research in somatic self-awareness ,20. The The cross-cultural adaptation of the FreSHAQ scale in Greek for patients with chronic shoulder pain was completed, presenting sufficient face and content validity. This was the first cross-cultural adaptation of this scale in another language and culture in relation to the original validation study. The results of the current study confirm that the scale possesses very good psychometric properties, comparable to the original scale, with very good internal consistency and excellent test–retest reliability. Regarding its construct validity, it correlates significantly with various indices of patient clinical status, and factor analysis revealed a one-factor solution for the FreSHAQ-GR, however, comprising the first six items of the original FreSHAQ-J scale. It is therefore recommended for further use in the clinical and research environment involving Greek-speaking patients with pain and functional limitations related to chronic shoulder pain conditions.


The growing incidence of myopia worldwide justifies the search for efficient methods of myopia prevention. Numerous pharmacological, optical, and lifestyle measures have already been utilized, but there remains a need to explore more practical and predictable methods for myopia control. This paper presents a review of the most recent studies on the prevention of myopia progression using defocus-incorporated multiple-segment spectacle lenses (DIMSsl), repeated low-level red-light (RLRL) therapy, and a combination of low-dose atropine (0.01%) with orthokeratology lenses. Myopia control is a topic of widespread discussion among ophthalmologists, optometrists, and other eye care specialists. There is a growing incidence of myopia observed worldwide. Considering geographical prevalence, myopia is particularly prevalent in industrialized East Asian regions . A meta-analysis conducted by Holden et al. estimated that, by the year 2050, nearly 50% of the global population will have myopia, with approximately 10% developing high myopia . Thus, pNumerous medical and environmental risk factors for myopia have been identified. The association between myopia prevalence and parental myopia is well documented . AdditioIn the pathomechanism of myopia, we understand that not only the axial length of the eye but also the structure of the anterior segment of the eye plays a significant role. Nevertheless, it is the axial length that is a key parameter for the progression of myopia . A largeStudies in the literature describe various methods for myopia control. A network meta-analysis by Huang et al.  lists seDefocus-incorporated multiple-segment spectacle lenses (DIMSsl) are designed to correct refractive errors while simultaneously preventing the progression of myopia. Each DIMSsl consists of a central zone for distance refractive correction and a surrounding zone comprising approximately 400 multiple defocus segments that create myopic defocus. Animal studies have demonstrated that imposed myopic defocus inhibits eye elongation, whereas hyperopic defocus promotes eye elongation .Repeated low-level red-light therapy involves using a device that emits low-level red light with a wavelength of 650+/−10 nm. The intervention requires looking into the device twice a day for 3 min, with a minimum 4-h interval between sessions. Parental supervision is necessary during the therapy .Orthokeratology and atropine eyedrops have separately demonstrated efficacy in slowing the progression of myopia . The mecThe current paper provides a review of the pertinent research on the prevention of myopia progression in children using three interventions: defocus-incorporated multiple-segment spectacle lenses (DIMSsl), repeated low-level red-light (RLRL) therapy, and a combination of low-dose atropine (0.01%) with orthokeratology lenses. We utilized the PubMed database, and only peer-reviewed clinical trials published in English between the years 2011 and 2023 were considered for inclusion .The first double-masked clinical trial assessing the efficacy of defocus-incorporated spectacle lenses (DIMSsl) was published in 2020 . The stuIn another publication from 2020,  documentThe visual function of the same group of patients was described in detail in a paper published in 2020 by Lam et al. . The stuIn 2022, Lam et al. published the results of a 3-year follow-up study . Their sp < 0.0001) and 30° nasal .In 2023, Zhang et al. published the results of the continued observation of changes in relative peripheral refraction associated with myopia progression . The stuGroup 1 (36 children) wore defocus-incorporated multiple-segment spectacle lenses (DIMSsl) for the entire 6-year duration;Group 2 (14 children) wore DIMSsl for the first 3.5 years and then switched to single-vision lenses (SVsl);Group 3 (22 children) wore SVsl for the first 2 years and then switched to DIMSsl for the remaining 4 years;Group 4 (18 children) wore SVsl for the first 2 years, then DIMSsl for 1.5 years, and finally switched back to SVsl for the last 2.5 years.A clinical trial conducted by Lam et al. and published in 2023 followed 90 Chinese children for a period of 6 years . The meaGroup 1: SER −3.04 ± 0.89 D/−3.69 ± 1.42 D;Group 2: SER −2.98 ± 1.13 D/−4.28 ± 1.15 D;Group 3: SER −2.68 ± 0.88 D/−3.92 ± 1.18 D;Group 4: SER −2.65 ± 1.18 D/−3.87 ± 1.53 D.The main measured outcomes were changes in axial length (AXL) and cycloplegic refraction. The spherical equivalent refraction (SER) at baseline and the 6-year follow-up for each group were as follows:Group 1: AXL 24.68 ± 0.76 mm/25.28 ± 0.81 mm;Group 2: AXL 25.00 ± 0.80 mm/25.71 ± 0.69 mm;Group 3: AXL 24.62 ± 0.79 mm/25.43 ± 1.01 mm;Group 4: AXL 24.42 ± 0.86 mm/25.14 ± 0.87 mm.The AXL measurements at baseline and the 6-year follow-up for each group were as follows:These results indicate that there was no rebound effect after stopping the use of DIMSsl.p < 0.001). In the second year, the spherical equivalent progression was −0.88 ± 0.62 D for DIMSsl and −1.23 ± 0.76 D for SVsl (p < 0.001). These findings provide further evidence of the effectiveness of DIMSsl in slowing myopia progression. A retrospective study conducted by , which aIn 2022, Jian Y. et al. published a multicenter, randomized, parallel-group, single-blind clinical trial . The stuIn 2023, Dong J. et al. conducted a double-masked clinical trial on repeated low-level red-light (RLRL) therapy involving 112 Chinese myopic children aged 7 to 12 years . The chin = 41), SVS-RLRL group (n = 10), RLRL-SVS group (n = 52), and RLRL-RLRL group (n = 11). Cycloplegic refraction and axial length were measured at the 24-month mark from the beginning of the RCT. Over the 2-year period, the mean change in axial length (AXL) and spherical equivalent refraction (SER) were found to be the smallest in the RLRL-RLRL group. However, a modest rebound effect was observed after the cessation of treatment.In 2022, Xiong R. et al. published a prospective, post-trial follow-up study or real-world study (RWS) [In a secondary analysis of data from a multicenter randomized controlled trial (RCT), Xiong R. et al. investigated the measurements of macular choroidal thickness (mCT) using swept-source optical coherence tomography (SS-OCT) and its associations with myopia control . The stuIn 2022, Chen Y. et al. conducted an RCT comparing the efficacy of repeated low-level red-light (RLRL) therapy with low-dose atropine for myopia control . The stuAnother RCT published in 2023 by Chen H. et al. analyzed the efficacy of low-intensity red light (LRL) therapy compared to single-focus spectacles (SFSs) for myopia control . The stuHe X. et al. conducted a study to assess the effectiveness of repeated low-level red-light (RLRL) therapy in children with premyopia, defined as a cycloplegic spherical equivalent refraction of −0.5 D to 0.5 D in the more myopic eye. The inclusion criteria also required at least one parent to have a spherical equivalent refraction of −3.0 diopters or less in either eye . The stuIn 2020, Tan Q. et al. reported the results of a one-year clinical trial that assessed the potential additive effect of 0.01% atropine eye drops in combination with orthokeratology . The stuIn 2023, Tan Q. et al. published the results of a two-year randomized controlled trial comparing the combination of orthokeratology with low-dose atropine to orthokeratology alone . The stuKinoshita et al. assessed the additive effect of orthokeratology and 0.01% atropine eye drops in myopia control in 41 Japanese children. In 2018, they published the results of the aforementioned clinical trial . The stuKinoshita et al. also conducted a two-year clinical trial to assess the efficacy of combined orthokeratology with 0.01% atropine in myopia control in 80 Japanese children aged 8–12 years old with spherical equivalent refraction (SER) ranging from −1.0 to −6.0 diopters . The parJiang J. et al. conducted a clinical trial to assess binocular and accommodative functions in children undergoing orthokeratology treatment combined with low-dose atropine . The stuZhao W. et al. described the effects of orthokeratology combined with atropine on choroidal thickness in a paper published in 2021 . The anaThe results of a clinical trial evaluating axial elongation and higher-order aberrations in children using orthokeratology combined with low-dose atropine were published in 2020 by Vincent SJ et al. . The stuA double-blinded randomized placebo-controlled trial evaluating the effectiveness of combined therapy with orthokeratology and low-dose atropine was conducted on 60 Chinese myopic children aged 8–12 years old . Thirty In this review, our focus was on the latest publications regarding myopia control. It is interesting to note that the oldest publication on the topic of “myopia control” available in the PubMed database dates back to 1933 and was published in The British Medical Journal by Sorsby . In thisThe quality of the trials described in this review is notable as they all measure myopia progression not only in terms of refraction but also by assessing the axial length of the eye. However, comparing the results accurately is challenging due to variations in materials and methods employed across the studies. While the participants in these trials are generally primary school children, the age range of participants differs among the studies. It would be valuable to assess the efficacy of the described methods in more specific age groups.All of the studies assess cycloplegic refraction, but different protocols and drops are used. For instance, Lam C.S.Y and colleagues used two drops of cyclopentolate 1% following one drop of Alcaine 0.5% and measured refraction after 30 min. They also verified the cycloplegic effect by measuring the amplitude of accommodation with the RAF rule . In a muAnother limitation of many studies on preventing the progression of myopia is the lack of a double-blinding process. Implementing the double-blinding process can be difficult and expensive for certain methods. For example, the only trial on RLRL therapy performed by Dong J. et al. was double-blinded due to the use of a sham device with reduced power .From a clinical perspective, it is important to consider not only effectiveness but also safety. Overall, all the described methods are considered safe, but they differ in invasiveness and time requirements. In the authors’ opinion, DIMSsl is the least invasive method, since most myopic patients are accustomed to wearing spectacles. Repeated low-level red-light therapy can be time-consuming. Orthokeratology users do not need to wear spectacles during the day, but they are required to wear contact lenses overnight. For many patients, managing contact lenses can be more burdensome than wearing spectacles during the day. Additionally, there is a risk of corneal complications associated with orthokeratology. Clearly, patients’ preferences and capabilities vary, and the optimal choice of therapy will depend on the individual needs of each patient.Currently, numerous options for slowing myopia progression are emerging, but further evidence is needed to support their efficacy. Many trials have relatively short observation periods, limiting our understanding of the long-term effects. Additionally, several trials are not double-blinded, particularly in the case of repeated low-level red-light therapy. It is crucial to conduct large-scale trials involving participants from different ethnic backgrounds to ensure the generalizability of the findings. Furthermore, assessing the long-term effects of different interventions, both in monotherapy and in combination, is necessary.Moreover, gaining a better understanding of the mechanisms of action underlying each method will provide a foundation for developing rational combined therapies. By unraveling the specific ways in which these interventions affect myopia progression, we can optimize treatment approaches and potentially enhance their effectiveness.


This study presents a method for a one-step co-encapsulation of PLGA nanoparticles in hydrophilic nanofibers. The aim is to effectively deliver the drug to the lesion site and achieve a longer release time. The celecoxib nanofiber membrane (Cel-NPs-NFs) was prepared by emulsion solvent evaporation and electrospinning with celecoxib as a model drug. By this method, nanodroplets of celecoxib PLGA are entrapped within polymer nanofibers during an electrospinning process. Moreover, Cel-NPs-NFs exhibited good mechanical strength and hydrophilicity, with a cumulative release of 67.74% for seven days, and the cell uptake at 0.5 h was 2.7 times higher than that of pure nanoparticles. Furthermore, pathological sections of the joint exhibited an apparent therapeutic effect on rat OA, and the drug was delivered effectively. According to the results, this solid matrix containing nanodroplets or nanoparticles could use hydrophilic materials as carriers to prolong drug release time. The course of OA is long and the disability rate is high. Joint replacement is often needed in the end stage, which causes pain and reduces patients’ quality of life ,2. The ppathways . In addipathways . Prolongpathways . The difOn the one hand, nanomaterials increase the relative molecular weight of free drugs, prolonging the residence time of drugs in joints. Nanoscale carriers can penetrate the extracellular matrix (ECM) and cell membrane, and release drugs directly into the ECM or into cells . Non-steIn this study, a drug delivery system using polylactic acid-hydroxyacetic acid copolymer (PLGA) nanoparticles and polyvinyl alcohol (PVA) was developed for intra-articular implantation of celecoxib nanoparticle fibrous membranes (Cel-NPs-NFs) by electrostatic spinning. The system was first prepared using PLGA and 2-HP-βCD as carriers by emulsifying solvent volatilization to produce drug-loaded nanoparticles (Cel-NPs). Next, the nanoparticles were mixed with a polymer solution and then encapsulated in nanofibers using electrostatic spinning technology. During this process the polymer solution mixed with nanoparticles is injected through a needle to produce a highly expanded and curved fluid jet and the dispersed phase tends to collect in the center of the fluid. The elongation effect of the fluid in the air helps the nanoparticles to settle inside the fibers rather than on the surface ,11. ThisPolyvinyl alcohol  was purchased from Corey International Trading Shanghai Co., Ltd. ; 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) was obtained from Zibo Qianhui Biotechnology Co., Ltd. ; Celecoxib was supplied by Tixiai Shanghai Huacheng Industrial Development Co., Ltd. ; Poly (lactic-co-glycolic acid) (PLGA) supplied by Jinan Jufukai Biotechnology Co., Ltd. ; DMEM, FBS, and PBS (pH = 7.4) buffer for cells was purchased from Gibco ; CCK8 enhanced solution supplied by Meilun Biotechnology Co., Ltd. ; Coumarin 6 was from Aladdin Reagent Shanghai Co., Ltd. ; DAPI Solution (1 mg/mL), dimethyl sulfoxide (cell culture grade) and trypsin-EDTA digest solution were obtained from Beijing Solarbio Science & Technology Co., Ltd. .w:w, 1:5) and dissolved in 5 mL of acetone and dichloromethane mixed solution  to form phase I. Then 20 mL of 1.5% HP-β-CD and 2% PVA aqueous solution formed phase II. Then phase I was injected slowly into phase II at 90 W ultrasonic power, 20kHz frequency, and phacoemulsification interval of 3 s for 30 min , followed by stirring at a low speed of 200 rpm  until the phase I organic solvent evaporated completely. Finally, distilled water was added until a volume of 20 mL was reached to obtain a 0.05% celecoxib nanoparticle solution (Cel-NPs).Celecoxib nanoparticles were prepared and optimized based on the emulsified solvent evaporation method . Brieflyw/v) PVA film-forming material  was added to the drug-containing nanoparticle solution until it was fully swollen and dissolved. Then the spinning solution was obtained. Next, with a voltage of 20 kV, injection rate of 0.7 mL/h, and a receiving distance of 140 mm, the uniaxial method  was adopted to spin the spinning solution with a high-voltage electrostatic spinning machine  and finally, a 3 × 5 mm film (Cel-NPs-NFs) was cut from the obtained drug-containing membrane.Using PVA as a membrane material, a celecoxib nanoparticle fiber membrane was prepared by electrospinning. First, 9% . Cel-NPs were directly diluted 10 times with water, and the emission scattering intensity was adjusted to  cps. Cel-NPs in Cel-NPs-NFs were measured by taking 0.1 g of Cel-NPs-NFs and dissolving it in 2 mL of water to prepare the sample solution; then, the emission scattering intensity was adjusted to  cps for measurement.The microscopic characterization of Cel-NPs and Cel-NPs-NFs was performed by transmission electron microscopy . Cel-NPs and Cel-NPs in Cel-NPs-NFs underwent a double dyeing method . Cel-NPs3) underwent low-speed centrifugation at 1000 rpm for 10 min, and Cel-NPs and a small amount of free drug was separated. Then, the free drug was deposited at the bottom; the upper liquid was a mixture of Cel-NPs and drug solution (C1). Secondly, the upper liquid was transferred into an ultrafiltration tube (molecular weight cutoff: 3k) and centrifuged at 4 °C and 13,000 rpm  for 30 min. Then, a small amount of free drug (C2) and Cel-NPs were separated. Finally, the encapsulation rate (EE%) of celecoxib in electrospun co-encapsulation of celecoxib NPs was determined according to Equation (1). The encapsulation rate of celecoxib in electrospun co-encapsulation of celecoxib NPs was determined by a high performance liquid chromatograph  ultrafiltration-assisted low-speed centrifugal method ,15. The The content of celecoxib in Cel-NPs solution was determined by HPLC (e2695 Waters HPLC with a Kromasil 100-5-C18 column (4.6 × 250 mm)), with mobile phase: the methanol-to-water ratio was 85:15, the injection volume was 20 μL, the flow rate was 1 mL/min, the column temperature was kept at 25 °C, and the detection wavelength was 254 nm). The Cel-NPs solution of 1 mL was transferred to a 10 mL volumetric flask, and an appropriate amount of methanol was added. After ultrasonic de-emulsification at 53 KHz for 30 min, the solution was filtered through a 0.22 µm microporous filter membrane, and the drug content was determined by HPLC.−1 at room temperature.IR  was used to distinguish the mode of action between celecoxib and carrier by taking appropriate amounts of celecoxib, Cel-NPs, and Cel-NPs-NFs. In the IR test, the dried sample and potassium bromide were uniformly ground evenly according to mass ratio of 1:100. Then, the tablets were scanned and analyzed in the wavelength range of 400–4000 cmDSC  was used to analyze the presence of the drug in the formulation. Then, 5 mg of celecoxib, Cel-NPs and Cel-NPs-NFs samples were weighed. The detection conditions of DSC were as follows: the heating rate was 10 °C /min, and the temperature range of the instrument was 30–300 °C.XRD  was used to further analyze the mode of action of celecoxib with the carrier. Celecoxib and Cel-NPs test state was powder, and Cel-NPs-NFs test state was a thin film. The parameters of the XRD measurement were as follows: the diffraction angle was 460°, the scanning step was 0.02°, the scanning mode was 2θ, the voltage of the Cu tube was 40 kV, and the current was 40 mA.Electrospun nanofibers have the advantages of a small diameter and a large specific surface area, and different membrane materials and drug loading methods affect the drug release rate at the drug delivery site. In this experiment, the membrane’s contact angle and mechanical properties were established to evaluate the process that could prepare the membrane. The contact angle can reflect changes in the wettability of the film surface: the smaller the contact angle, the better the wettability . The sur0 is the total amount of drug injected and released, Ct is the drug concentration at time point t (ug/mL), Ci is the drug concentration measured at a time point before the t time point (ug/mL), V is the sampling volume (2 mL).The in vitro release behavior of Cel-NPs and Cel-NPs-NFs were evaluated by the dialysis bag method ,19,20, s5 cells per well) for 24 h while the cells grew to 80%. C6-NPs and C6-Nps-NFs containing 0.18 µg coumarins were cultured in serum-free medium and incubated for 0.5 h and 4 h, respectively, without light at 37 °C. The supernatant was discarded, and the cells were fixed in 4% paraformaldehyde solution at room temperature for 15 min, washed with cold PBS three times, and then observed under a fluorescence confocal microscope . The density of 2 × 105 cells per well was quantitatively analyzed. After the above steps, until the end of uptake and trypsin digestion, the cells were collected by 3 × 103 r/min, centrifuged for 5 min, and resuscitated with 0.5 mL PBS. The average fluorescence intensity (MFI) of 10,000 cells was detected by flow cytometry .Coumarin is a substance with similar solubility to celecoxib and can be used as a probe for fluorescence labeling. The nanoparticle fiber membrane (C6-NPs-NFs) co-encapsulating coumarin-6 (C6-NPs) was prepared by the same method, and its particle size potential was determined by nanocarrier size, polydispersity, and particle charge methods to study the uptake of the preparation in mouse macrophage RAW 264.7  Cell item number was PWE-MU004-2). The CCK8 method was used to determine the cytotoxicity of blank nano-carrier solution and nano-fiber spinning solution when the concentration of celecoxib was a gradient of 125–15,625 µg/mL. RAW264.7 was cultured in a 12-well plate (1 × 10n = 8). The positive control group was given 10.5 mg/kg celecoxib capsules by intragastric administration once a day. In contrast, in the Cel-NPs-NFs treatment group, the nanoparticles were implanted in the articular cavity (drug content: 20 µg). Seven days later, the rats were intraperitoneally injected with 2% pentobarbital sodium and sacrificed. The skin and subcutaneous tissue were separated from the knee joint, and the surrounding muscle tissue was removed. The whole knee joint of rats was amputated, preserving a quarter of the femur and tibia. The injury to cartilage was observed by photography, and the gross cartilage was scored by the Pelletier scoring standard [The animal model of osteoarthritis (OA) established by injecting sodium iodoacetate into the knee joint is a classical and reliable model to study osteoarthritis ,22. The standard . The knestandard .X ± SD. Pairwise comparisons were made by the Welch-t test. Dunnett’s multiple comparison tests were used for comparison between multiple groups. The tests revealed no significant differences when p > 0.05.The data were collected and analyzed. GraphPad prism 8.0.2 was used for statistical analysis of the experimental data and expressed by Nanoparticles can increase the amount of drugs delivered into inflammatory synovial tissues and are more advantageous than microspheres and other particle drug delivery systems . The copIn this study, celecoxib was first prepared as Cel-NPs. Then Cel-NPs-NFs were prepared, combining the advantages of NFs and NPs, to provide a reference for implanted drug administration of arthritis. There was no apparent change in the morphology of Cel-NPs before and after electrospinning. Cel-NPs before and after electrospinning exhibited a spherical shape a. The avThrough the tensile test, we can judge the change in the stress–strain curve of the film in the elastic stage, yield stage, strengthening stage, and local deformation stage g. The YoAs shown in −1 and 3342 cm−1 is the characteristic peak of celecoxib. The characteristic peak of S=O2 could be seen at 1402 cm−1 and 1146 cm−1  by flow cytometry. There was no significant difference in intake between the NPs and NPs-NFs groups at 4 h (p > 0.05)  d. The lap < 0.05) and the Cel-NPs-NFs treatment groups (p < 0.001) compared with the model group. The rat hind limb grip measurement a and behp < 0.0001), and there was a more significant difference in the Cel-NPs-NFs treatment group (p < 0.05). The overall results showed that the significant difference in the Cel-NPs-NFs treatment group was more robust. A more likely reason may be that the administration mode of the Cel-NPs-NFs treatment group reached the cartilage surface directly without going through the systemic circulation, NPs could be retained on its surface for a long time because of its nature, and the NFs carrier also had a good retention lubrication effect. On the one hand, it could form a drug store in the joint cavity and release NPs for a long time, effectively inhibiting the cartilage damage caused by inflammation. Its lubrication effect could reduce the cartilage friction caused by its daily activities, with a double protective effect.Hematoxylin–eosin staining showed that in the normal group, the cartilage structure was intact d, there Using PVA as the spinning material, Cel-NPs-NFs were successfully prepared by loading stable NPs on the fiber membrane by electrospinning. Celecoxib was found on the fiber membranes in an amorphous state, with good resistance and hydrophilicity. The in vitro release test showed that its release ability was sustained for seven days due to the secondary encapsulation in the fiber membrane, and its release behavior changed from Fick diffusion to the joint action of Fick diffusion and skeleton dissolution. The in vitro uptake experiment showed that compared with the NPs and NPs-NFs systems, the cell uptake efficiency significantly improved in a short time. The in vitro experiments showed that the NPs-NFs system was beneficial in prolonging the drug release time and improving the cellular uptake efficiency. Furthermore, in the study of the rat arthritis model, the gross cartilage and hematoxylin–eosin staining after treatment showed that Cel-NPs-NFs had a better therapeutic effect than oral administration. To summarize, NPs-loaded NFs as a drug delivery system have great potential in the field of controlled drug release research.


Lithium-sulfur technology is a strong candidate for the future generation of batteries due to its high specific capacity (1675 mAh g In the past two decades, the demand for energy produced through renewable sources has continuously increased, which has limited fossil fuel utilization and related COmissions . Therefomissions ,4. Howevmissions . Future missions ,7, the amissions , or the missions  are undelow cost ,11,12. Hthe cell ,14. The When the cell is discharged, the redox reaction forms lithium polysulfides (LiPSs) intermediates, consisting of Li process . The piv process ,17. Up t process . One app process ,20. Howe process ,22,23. A process ,25, N-do process , and gra process  with dif process ,29 or me process ,31. More of Li2S . Thus, t of Li2S . In this of Li2S  designed of Li2S  introducIn this work, we prepared an innovative composite material based on a nitrogen-sulfur-doped graphene matrix on which zinc sulfide nanoparticles were directly nucleated in a one-step microwave synthesis. The combination of the two materials turns out to be a winning strategy for obtaining a cathode material suitable for lithium-sulfur batteries: the graphitic network can provide good electronic conductivity for the conversion of the sulfur, while the graphene doping and the presence of ZnS nanoparticles guarantee a strong interaction and catalytic activity towards LiPSs, thus alleviating the shuttle effect.The graphene oxide (GO) was purchased from Cheaptubes, HAll the chemicals were used as purchased without further purification. The sulfur-nitrogen doped rGO (labelled as SN-rGO) and the sulfur-nitrogen doped rGO embedded with ZnS nanoparticles, labelled as SN-rGO/ZnS), were prepared by following the synthesis procedure reported below. With regards to the SN-rGO sample, in a microwave 100 mL Teflon reactor equipped with pressure and temperature probes , 105 mg of GO  were added to 50 mL DI water and sonicated (Elmasonic P 30H) for 30 min. After that, 71 mg of HThe same procedure was followed for both SN-rGO and SN-rGO/ZnS samples. Pure sulfur (≥99.5% Sigma Aldrich) and the SN-rGO (and SN-rGO/ZnS) powders were gently mixed using a pestle and mortar in a weight ratio of 3:1. The mixture was then transferred to a furnace tube under argon stream, where the material was heated with a hitting rate of 2.5 °C/min to 155 °C for 12 h, in order to obtain the infiltration of the sulfur in the carbonaceous material. The melt-infused composites, referred as SN-rGO/SThe cathodes were obtained through the solvent tape casting method. The SN-rGO/SXRD analysis was carried out by a PANalytical X’Pert (Cu Ka radiation) diffractometer. Data were collected with a 2D solid state detector (PIXcel) from 10 to 80°2ϑ with a step size of 0.026°2ϑ and a wavelength of 1.54187 Å. The Brunauer-Emmett-Teller specific surface area (SSA) was determined by nitrogen physisorption at 77 °K using a Micrometrics ASAP 2020 instrument. The specific surface area was calculated with the BET model in the relative pressure range of 0.07–0.30 by assuming 0.162 nmThe UV-vis absorption spectra were detected by a UV–vis spectrophotometer  within the spectral range of 300–550 nm.To measure the electrochemical properties of the SN-rGO/Sv/v) with 1.0 M lithium bis(trifluoromethanesulfonyl)imide  and 1,3-dioxolane (DIOX) 1:1  using an Arbin LBT-21084 battery tester at room temperature. Galvanostatic discharge-charge tests were carried out in the potential interval 1.85–2.6 V vs. Li/Liv/v) Liv/v).Liaterials . In partv/v) LiThe Tafel and the LiThe microwave synthesis approach that was used in this work to obtain the SN-rGO and the SN-rGO-ZnS is illustrated in ve sites ,38. With process ,40. Concal route .The morphological characterization of SN-rGO and SN-rGO/ZnS was performed by FESEM technique, as reported in The functional groups and surface chemical composition of the SN-rGO and SN-rGO/ZnS were investigated by means of X-ray photoelectron spectroscopy (XPS) as shown in -rGO/ZnS e. The otgraphene . The N1sgraphene ,49,50. TThe characterization of the pristine SN-rGO and SN-rGO/ZnS was completed utilizing XRD, BET and TGA techniques, which were also used to study the two materials after they had been loaded with sulfur in the tubular oven . The inf 0.38 nm . FinallyThe nitrogen adsorption-desorption analysis of SN-rGO and SN-rGO/ZnS, shown in e latter . The TGASince one of the fundamental characteristics of cathode material in lithium-sulfur batteries is to have a good interaction and enhanced catalytic activity to accomplish a faster kinetic reaction of conversion of LiPSs to reduce the shuttle effect, we focused the first electrochemical tests on investigating our samples in this regard. In particular, the adsorption capability of our materials  towards LiPSs was evaluated by dipping 30 mg of powders in 4 mL of DME:DIOX 1:1 mixture LiIn order to verify the role played by the doping process and ZnS nanoparticles in the kinetic reaction of LiPSs conversion, we performed cyclic voltammetry (CV) tests in the voltage window −1 ∼ 1 V of symmetrical cells with both a catholyte Lilyte see , as repoal cells  are charThe redox kinetics of our samples during the discharge and charge process was investigated by current exchange measurements using linear sweep voltammetry (LSV) with a LiAnother essential aspect that must be considered in lithium-sulfur batteries is the liquid-to-solid conversion process of long polysulphides in Li surface . Afterwa surface . Going iTo exhaustively study how the morphology of our samples and the ZnS nanoparticles affect the kinetics features of Liwth rate ,65. Thesactivity , thus av2 over the baseline current. A more detailed discussion about the determination of the onset potential is reported in In order to investigate the difference in electrochemical performance of Li–S cells based on different cathodes, coin-cell batteries with a sulfur loading of 1.1–1.3 mg cmly to S8 ,67,68. Frode see , it was By increasing the scan rate of the CV, it was possible to investigate the lithium diffusion into the electrodes by using the Randles-Sevcik equation (Equation ), accord cathode . The latThe electrochemical impedance spectra (EIS) of the fresh cell and after each cycle of the CV at different scan rate was performed to get more information about the kinetics of the reactions occurring in our cells. As shown in be noted , with thTo evaluate the electrochemical performance of our cathodes, we performed a long cycling in the potential window of 1.85–2.6 V vs. Li/Liproposed . Anothermulation ,72,73. TFinally, the rate performance at increasing current density of the two different cathodic formulations was tested, as shown in Moreover, as This work reports a facile and fast microwave-assisted synthesis of a composite material SN-rGO/ZnS as a sulfur host for high-performance Li-S batteries. In particular, the proposed production method can significantly reduce the synthesis times compared to a classic hydrothermal process , ensuring the high reproducibility of the materials. The peculiarity of our cathode lies in the combination of a material with high electronic conductivity (sulfur-nitrogen doped rGO), which is required to have good use of sulfur, with a material (ZnS nanoparticles) that exhibits good affinity towards LiPSs and can catalyze the reaction of conversion from SThe chemical-physical characterization of our samples confirms, through XPS, FESEM, and XRD analysis, the heteroatomic (sulfur and nitrogen) doping process of graphene and the formation of ZnS nanoparticles on its surface. As reported in the literature, pyridinic nitrogen and ZnS nanoparticles are important factors since they are the main sites that can strongly interact with LiPSs.The electrochemical characterization consisting of cyclic voltammetry, EIS, Li


Background: The tumor microenvironment (TME) in cholangiocarcinoma (CCA) influences the immune environment. Checkpoint blockade is promising, but reliable biomarkers to predict response to treatment are still lacking. Materials and Methods: The levels of checkpoint molecules , macrophages (CD68), and T cells (CD4 and CD8 cells) were assessed by multiplexed immunofluorescence in 50 intrahepatic cases. Associations between marker expression, immune cells, and region of expression were studied in the annotated regions of tumor, interface, sclerotic tumor, and tumor-free tissue. Results: ICCA demonstrated CD4_TIM-3 high densities in the tumor region of interest (ROI) compared to the interface (p = 0.014). CD8_PD-L1 and CD8_ICOS densities were elevated in the sclerotic tumor compared to the interface . In a multivariate model, high expression of CD8_PD-L2 (p = 0.048) and CD4_ICOS_TIGIT (p = 0.011) was associated with nodal metastases. Conclusions: High densities of PD-L1 were more abundant in the sclerotic tumor region; this is meaningful for the stratification of immunotherapy. Lymph node metastasis correlates with CD4_ICOS_TIGIT co-expression and CD8_PD-L2 expression, indicating the checkpoint expression profile of patients with a poor prognosis. Also, multiple co-expressions occur, and this potentially suggests a role for combination therapy with different immune checkpoint targets than just PD-1 blockade monotherapy. Cholangiocarcinoma (CCA) is a deadly disease occurring in the liver and is associated with high mortality rates . This caIntrahepatic cholangiocarcinoma (iCCA) demonstrates phenotypic differences in immune cell infiltrate, genetics, and stroma . Recent In the TME, there is a homeostasis favoring either the host immune response or the tumor growth . Here, cGood biomarkers to predict response to ICIs are still lacking, and even with high expression levels of PD-L1, response rates vary between 37% for combination chemotherapy and immunotherapy  and 5.8%In this study, we have integrated the co-inhibitory checkpoints TIM3, LAG3, CTLA-4, TIGIT, PD-1, PD-L1, PD-L2, and the co-stimulatory checkpoint ICOS. These checkpoint molecules are all in advanced stages of clinical development, and some therapeutic targets are available or in development. The inhibitory checkpoints are regulators of the immune system. PD-L2 is a second ligand for PD-1 and inhibits T-cell activation, just as PD-L1. The PD-1 receptor is expressed on the surface of activated T cells. PD-1 and PD-L1/PD-L2 belong to the family of immune checkpoint proteins that act as co-inhibitory factors that can limit the development of the T cell response. PD-L1 and PD-L2 expressed on the tumor cells bind to PD-1 receptors on the activated T cells, which leads to the inhibition of the cytotoxic T cells. These deactivated T cells remain inhibited in the tumor microenvironment.Additionally, we analyzed the presence of the immune cells CD4, CD8, and CD68 in primary resected patients with iCCA using multiplexed imaging on whole slide imaging. Our aim was to perform deep immunophenotyping and characterize the immune environment and expression of immune checkpoint differences between ROIs  slides were collected, and the slide with the vital tumor and the presence of the interface area was selected by the pathologist (LH). The selected block was used for further processing for our multiplexed imaging workflow using the Tissuefaxs method . For tumor staging and grading, the TNM classification was used according to the AJCC/UICC 8th edition.All FFPE samples were subjected to multiplexed immunofluorescence (mIF) in serial 5 μm histological tumor sections obtained from representative FFPE tumor blocks. The FFPE blocks were carefully selected for the presence of the tumor region and, if available, the interface region. The sections were labeled using the Opal 7-Color fIHC Kit . The antibody fluorophores were grouped into a panel of 5 antibodies. The order of antibody staining was always kept constant on all sections, and sections were initially counterstained with DAPI (Vector Laboratories). The multiplexed immunofluorescence panel consisted of CD4, CD8, CD68, PD-1, PD-L1, PD-L2, ICOS, TIGIT, TIM-3, CTLA-4, and LAG-3 See . All ant® HardSet™ Antifade mounting medium.The manual for mIF is described as Edwin R. Parra’s protocol : in shorThe slides were then digitally scanned with the TissueFAXS PLUS system . Image analysis was performed in different regions of interest (ROI) in each image (only if present in the slide): tumor, tumor-free, interface, and sclerotic tumor. The size of the ROI varies per slide. Immune cell expression was calculated in percentages throughout the whole project.2 using cell segmentation, and thresholds were set manually under the supervision of two pathologists (LH/MD). Positive cell counts were categorized based on thresholds; a value above the threshold was considered positive. Checks were performed by the pathologists (LH/MD).Strataquest software was used to analyze the antibody staining and cell counts. The library information was used to associate each fluorochrome component with a mIF marker. All immune cell populations were quantified as positive cells per mmThe primary endpoint of this study was to compare immune cell composition, distribution, and co-expression of checkpoints in iCCA. Group comparison was performed on the tumor and interface regions. The tumor ROI was outlined in areas with vital cancer cells, avoiding areas with necrosis. Sclerotic tumor was defined in areas with the presence of dense eosinophilic, sclerotic stroma, usually located in the central area of the tumor only. The interface area was outlined in cases with the presence of the invasive margin of the tumor, usually in the area where the cancer cells invade the surrounding tissue. Tumor-free ROI was outlined in slides with the presence of cancer-free tissue.The different ROIs were compared with the aim of visualizing distribution differences within one slide. Associations of immune cell subsets with clinical variables were investigated by means of binary logistic regressions. Therefore, immune cell expression data was converted into a dummy variable (high expression vs. low expression) using the median expression as a cut-off for grouping between high and low expression.t-test was applied to determine statistically significant differences between values of immune cells within ROIs. The level of significance was set to alpha = 0.05, and p-values were calculated using 2-sided testing. All statistical calculations were implemented using Python .Group comparisons were conducted by the Mann–Whitney U test or T-test in the case of continuous variables, while the χ2 test and Fisher’s exact test in accordance with the scale and number count were used in the case of categorical variables. The Wilcoxon matched-pairs test or the paired The clinical cohort comprised 50 patients with localized iCCA that underwent curative-intent surgery at our hepatobiliary department. Due to our aggressive department standards, a notable number of patients presented with multifocal disease (34%). A subset of patients also underwent neoadjuvant therapy (6%). Major liver resection was the treatment of choice in the majority of the patients (86%), resulting in an R0 resection rate of 92%. Nodal metastases were present in 32% of the overall cohort. Further pathological examination revealed T2 tumors to be the most prevalent (48%) among the patients. The median recurrence-free survival (RFS) of the cohort was 8 months, while the median overall survival (OS) was 32 months. More details regarding the clinical characteristics of the cohort are presented in 2.We first analyzed the immune environment and spatiality; the outlined ROIs were used for comparison: tumor, sclerotic tumor, interface, and tumor-free tissue. The ROIs differed in size; positive cell counting was performed and measured in percentage per mmp = 0.034 and p = 0.0002, respectively). The interface compartment was mainly infiltrated with CD8-positive T cells when compared to tumor and sclerotic tumor ROI . There was no significant difference in the distribution of CD68 between the different ROIs.The CD4 and CD8 T cells were more abundant in the tumor area compared to tumor-free tissue (p = 0.02). CD4 with co-expression of TIM-3 was more abundant in the tumor compared to interface (p = 0.0137), just as was CD4 with PD-1 and TIM-3 co-expression (p = 0.027).The comparison of co-expression of checkpoint molecules between the different ROIs illustrated higher levels of CD4 with CTLA-4 in the sclerotic tumor compared to the interface (p = 0.0309 and p = 0.0112 respectively). Moreover, CD8 with PD-1 and PD-L1 and CD8 with PD-1 and PD-L2 co-expression were more abundant in the sclerotic tumor . See p-values between the ROIs in iCCA.CD8 cells with co-expression were significantly increased in the sclerotic tumor when compared to the tumor. CD8 with ICOS and PD-L1 positive cells were more abundant in the sclerotic tumor (p = 0.041), CD8_PDL2 (p = 0.041), CD4_ICOS_TIGIT (p = 0.024), CD4_TIGIT (p = 0.015), CD8_ICOS_TIGIT (p = 0.015), and CD8_ICOS_CTLA (p = 0.015) within the tumor and low expression of CD8_CTLA (p = 0.026) and CD8_TIM3 (p = 0.026) within the normal liver tissue were associated with nodal metastases in iCCA. In a multivariate model, high expression of CD8_PDL2 (p = 0.048) and CD4_ICOS_TIGIT (p = 0.011) were identified as the two independent predictors of nodal metastases in iCCA. More details are depicted in To investigate associations between immune cell subsets and the presence of lymph node metastases, we conducted binary logistic regressions. In iCCA high expression of CD8_PDL1_PDL2 (p-value < 0.1 were transferred into a multivariate model using backward elimination. No multivariate analysis was conducted for perihilar cholangiocarcinoma as only tumor_CD68 was associated with lymph node metastases. For intrahepatic cholangiocarcinoma the following variables were included in the multivariate model: Tumor_CD8_PD-L1_PD-L2, Tumor_CD8_PD-L2, Tumor_CD4, Tumor_CD4_ICOS_TIGIT, Tumor_CD4_TIGIT, Tumor_CD4_TIGIT_CTLA-4, Tumor_CD8_ICOS, Tumor_CD8_ICOS_TIGIT, Tumor_CD8_ICOS_CTLA-4, Liver_CD8_PD-1_PD-L1, Liver_CD68_PD-1_PD-L1, Liver_CD4_CTLA-4, Liver_CD8_CTLA-4, Liver_CD4_TIM-3, Liver_CD8_TIM-3. OR, Odds ratio.Various Immune cell subsets were prognostic for the presence of nodal metastases. All immune cell subsets within the tumor, interface and normal liver were investigated but, only Immune cell subsets with a significant association in univariate and multivariate analysis are depicted within the table. Immune cell subsets displaying a Cholangiocarcinoma is a heterogeneous disease, and therapeutic targets are still evolving. The response and mechanisms to resistance to immunotherapy are likely to be found in the TME, but a lot is still unknown, and good biomarkers to predict which patients will respond are still lacking. Intratumoral genetic heterogeneity plays an important role in the response to treatment , and theHere, we investigated the immune cell composition and identified differences in immune checkpoint molecule expression between different ROIs, within the same histology slide. The CD8 cells are most highly expressed in the interface area, the invasive margin for the cancer cells to grow into the surrounding tissue, and here their contribution to supporting the host immune system is needed. There was no significant difference in abundance between the ROIs. An explanation for this could be that macrophages are present in large numbers in normal livers as well. CD68 will also stain the resident Kupffer cells. Macrophages can be recruited to areas with damage, such as inflammation or necrosis, but not all macrophages stained by CD68 are tumor-associated macrophages.The checkpoint molecules are mostly expressed in the sclerotic tumor and the tumor, suggesting immune checkpoint therapy could have a possible effect here by releasing the blocked CD4 and CD8 cells. The CD4 T cells demonstrated expression of the immune checkpoint molecules TIM-3 and PDL-1, both with an immunosuppressive effect in the tumor and interface ROI. The CD8 T cells expressed PD-1, PD-L1, and PD-L2, all having an immunosuppressive effect as well. Further CD8 T cells with co-expression of ICOS were significantly more present in the sclerotic tumor. The sclerotic tumor lesion is expected to be the older central core of the lesion. ICOS has an immune stimulatory effect, and patients with low levels of ICOS might demonstrate a good response to treatment strategies where the immune anti-tumor response is stimulated . CombineThe co-expressions of the checkpoint molecules on the T cells indicate an immunosuppressive environment in this tumor entity. The checkpoint phenotype demonstrated more PD-1, ICOS, and TIM-3 checkpoint expression on CD8 cells. The fact that the interface is mostly lacking in the expression of the checkpoint molecules is an important finding since this is the area where the cancer cells are most aggressive and invade the surrounding tissue. In order to stop the tumor from growing, the interface is an important target area from a biological point of view. Moreover, the presence of effective immune cells in the surrounding regions of the tumor is an important finding. Further research is needed to investigate if immune cells can be recruited to the interface area when the immune cells are released by ICI therapy.The zonation in the distribution of the checkpoint molecules could be of importance in deciding to treat patients with a combination of chemotherapy and immunotherapy. Currently, PD-L1 expression is still used as a marker to predict response to immunotherapy. When the CPS score is used for patient stratification and this is done on a biopsy in an advanced setting, potentially, patients get unjustly rejected for the treatment. This is of primary importance, while the use of immunotherapy, either in combination or alone, is justified in an advanced setting when other therapeutic strategies are no longer an option. Our data show that the PD-L1 expression on immune cells is highest in the sclerotic tumor area, usually the older central core of the tumor. It is questionable if this region is sufficiently represented in a core biopsy. See Multivariate analysis identified CD8_PD-L2 and CD4_ICOS_TIGIT to be related to nodal metastases in iCCA. Another group has identified CD8 and Tregs as being associated with poor outcomes and nodal metastases . Our finAnother important finding is that expression of multiple checkpoint combinations, not just the overexpression of PD-1, was observed. This finding suggests a different therapeutic approach since this tumor will be PD-1 blockade refractory when treated with PD-1 blockade therapy alone. New generations of immune checkpoints, such as TIGIT and ICOS, are expressed as well. Targeting iCCA could mean that a combination of different immune checkpoint agents is a better approach than just a PD-1 blockade monotherapy. It is of primary importance that these new therapeutic strategies are being tested, potentially in a combination immunotherapy and chemotherapy setting. Further research is needed to validate our results.


Currently, the global trend of several hundred thousand new confirmed COVID-19 patients per day has not abated significantly. Serological antibody detection has become an important tool for the self-screening of people. While the most commonly used colorimetric lateral flow immunoassay (LFIA) methods for the detection of COVID-19 antibodies are limited by low sensitivity and a lack of quantification ability. This leads to poor accuracy in the screening of early COVID-19 patients. Therefore, it is necessary to develop an accurate and sensitive autonomous antibody detection technique that will effectively reduce the COVID-19 infection rate. Here, we developed a three-line LFIA immunoassay based on polydopamine (PDA) nanoparticles for COVID-19 IgG and IgM antibodies detection to determine the degree of infection. The PDA-based three-line LFIA has a detection limit of 1.51 and 2.34 ng/mL for IgM and IgG, respectively. This assay reveals a good linearity for both IgM and IgG antibodies detection and is also able to achieve quantitative detection by measuring the optical density of test lines. In comparison, the commercial AuNP-based LFIA showed worse quantification results than the developed PDA-based LFIA for low-concentration COVID-19 antibody samples, making it difficult to distinguish between negative and positive samples. Therefore, the developed PDA-based three-line LFIA platform has the accurate quantitative capability and high sensitivity, which could be a powerful tool for the large-scale self-screening of people. The global outbreak and rapid spread of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused serious damage to the global economy and posed a serious threat to global public health security . Early dCurrently, many detection techniques have been developed for COVID-19, among which nucleic acid amplification tests and immunoassays are the main tools for the clinical diagnosis of infections . They haThe ideal autonomous immunoassay should be inexpensive, rapid, easy to perform, and have high accuracy . To obtaTo address the above issues, we developed a three-line LFIA immunoassay based on polydopamine (PDA) nanoparticles for the accurate and rapid detection of IgG and IgM. In addition, the developed colorimetric LFIA showed a higher sensitivity for antibody quantification in serum samples than the commercial AuNP-based LFIA. Therefore, we believe that the developed PDA-based three-line LFIA colorimetric platform with accurate quantification capability and high sensitivity can be a powerful tool for low-cost, rapid, and accurate immunoassays for the large-scale screening of COVID-19.PEG 2000) was purchased from Yusi Co., Ltd. . Dopamine hydrochloride, tetraethyl orthosilicate (TEOS), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), triton X-100, tween 20, and trizma base were purchased from Sigma-Aldrich . Sodium polystyrene sulfonate  was purchased from Shanghai Zhenjun Biotechnology Co., Ltd. . Poly  and polyvinylpyrrolidone  were purchased from Shanghai Maclean Biochemical Technology Co., Ltd. . Ammonia solution (25%) was purchased from Tianjin Beilian Chemical Co., Ltd. . Sucrose was obtained from Tianjin Hedong District Chemical Reagent Co., Ltd. . Sodium chloride was purchased from Tianjin Tianli Chemical Reagent Co., Ltd. . Ethanol, methanol, and isopropanol were all provided by Tianjin Fuyu Fine Chemical Co., Ltd. . Sulfo-NHS was provided by Shanghai Yanchang Biochemical Technology Development Co., Ltd. . MES and HEPES were purchased from MP Biomedical. . Glycine was purchased from Biotech Bioengineering Co., Ltd. . SARS-CoV-2RBD antigen, mouse IgG, and goat anti-mouse IgG were all provided by Beijing Berseth Biotechnology Co., Ltd. . COOH-PEG-Silane  was then rapidly added to the solution and stirred for 24 h. The solution was mixed with acetone at a ratio of 1:2 and left to settle for 24 h. The supernatant was removed by centrifugation, and the precipitate was collected . The precipitate was washed three times with acetone and then redispersed in deionized water and stored at 4 °C.2 was centrifuged  three times and redispersed in methanol.5 mL of polydopamine nanoparticles (PDA NPs) were added to 5 mL of a 2 g/L aqueous PSS solution with 6 mM NaCl and stirred vigorously for 3 h. The solution was centrifuged twice continuously  to remove excess magazines and resuspended in 5 mL of deionized water. Then, the PSS-modified PDA was added dropwise to 5 mL of a 2 g/L PAH aqueous solution containing 6 mM NaCl, stirred vigorously for 3 h, centrifuged twice continuously , and the solution was resuspended in 5 mL of deionized water. Next, 5 mL of PAH-modified PDA was added to 5 mL of a 4 g/L PVP solution and stirred overnight. The PVP-modified PDA was centrifuged  to obtain a precipitate that was dispersed in 0.2 μL of deionized water. After adding 1 mL of isopropanol and 0.46 mL of water with vigorous stirring, 1.43 mL of ammonia-isopropanol solution (3.84 vol%) was added to the system. 0.4 mL of TEOS-isopropanol (0.97 vol%) was added to the solution with gentle stirring, and the reaction was carried out for 2 h. PDA@SiO2 with vigorous stirring for 2 h. After that, the solution was centrifuged at 8000 rpm for 10 min at 25 °C and then washed with methanol and deionized water twice. Finally, the PDA@PEG was dispersed with deionized water.COOH-PEG-silane was dissolved in methanol  and then added to 950 μL of PDA@SiOv/v DMSO, 20 mM MES) and resuspended in 150 μL of DMSO-MES buffer, and the solution was sonicated to a homogeneous photodispersion. 6 μL of 6 mg/mL EDC and 6 μL of 6 mg/mL NHS were added to the solution and sonicated continuously for 30 min. Next, 800 μL 20 mM MES buffer and 37.5 μL different concentration ratios of SARS-CoV-2 RBD antigen and mouse IgG solution  were sequentially added to the system and incubated at 37 °C with 100 r/min in a shaker for 2 h. Finally, 30 μL of glycine stop buffer was added to the system and the resulting PDA-antigen complexes were washed with storage buffer , and the complexes were redispersed in storage buffer and stored at 4 °C.200 μL of PDA@PEG solution was washed three times with 200 μL of DMSO-MES buffer  at an accelerating voltage of 200 kV. A Zetasizer Nano ZSE was used to determine the dynamic light scattering (DLS) and Zeta potential of PDA nanoparticles. A Nikon D90 camera was used to take images of PDA-based LFLA and AuNP-based LFIA. All measurements were performed under uniform room-temperature conditions.0.5 μL of 1 mg/mL SARS-CoV-2 anti-human IgG and 0.5 μL of 1 mg/mL SARS-CoV-2 anti-human IgM were immobilized on NC membranes for forming two test lines, and 0.5 μL 0.2 mg/mL goat anti-mouse IgG was immobilized on a NC membrane to form a control line. The prepared LFIA was placed in a drying oven to be dried at 37 °C for 2 h and then stored at 25 °C for further use.A series of standard solutions  were prepared by stepwise dilution of IgG and IgM produced against the S protein and mixed with human serum. Next, 30 µL of the standard antibody solution was thoroughly mixed with the assay solution, which contained 10 µL of PDA probe solution and 60 µL of HSLF buffer . The solutions to be tested were added to LFIA for IgG and IgM detection. For the AuNP-based commercial LFIA , 30 µL of antibody solution was mixed by dilution with 60 µL of commercial buffer to perform the test. 15 min later, the results of the LFIA were captured by a Nikon D90 camera under the same conditions. Image J was used to measure the optical density values of the test line (T) and the control line (C), with the antibody concentration reflected by the grey value of the T line.The software for automatic quantification was developed using Python and the graphical user interface development framework PyQt5. The software contains two main parts. The first part is the image reading part, which is made up of an image read button and an image viewer. After pressing the image read button, users can read images from local storage and show the images on the image viewer. On the image viewer, users can choose the test line region. The second part is the quantification part. This part is composed of two select buttons for deciding the type of strip (IgG or IgM), a quantification button, and a result viewer. With the test line region and strip type selected, users can press the quantification button to show the concentration of the strip on the result viewer.Since the detection of IgM and IgG can provide information on the time course of viral infection, rapid detection of IgM and IgG can support the diagnosis and treatment of COVID-19 disease . To achiPolydopamine is one kind of strongly adhesive polymer that is simple to prepare, environmentally friendly, and low cost . Benefity = 7.16084x + 97.0.5087 (R2 = 0.99936) and IgG: y = 7.1867x + 97.13737 (R2 = 0.9951), as shown in To evaluate the sensitivity of IgG and IgM antibodies detection by PDA-based three-line LFIA, we spiked serum samples with different concentrations of COVID-19 IgM and IgG antibodies. Here, we simulated the samples with different antibody concentrations of COVD-19 patients by mixing IgM and IgG in serum, respectively, while setting a concentration gradient for IgM or IgG antibodies for detection . The detection results were taken under the same environmental conditions to be read out. As shown in 0 represents the gray value of T lines in the negative group , and T represents the gray value of T lines in the positive group . Therefore, the PDA-based LFIA was proven to have a better quantitative capability for serum samples compared with the AuNP-based commercial LFIA. Importantly, both methods require only 15 min for the detection process. Thus, the PDA-based three-line LFIA is more advantageous in terms of accuracy. However, the detection of IgG and IgM-based antibodies requires the collection of a serum sample, which can be uncomfortable for users and may lower the test’s overall acceptability.To evaluate the accuracy of PDA-based three-line LFIA detection better, we compared the detection performance of PDA-based LFIA and the most widely used commercial AuNP-based LFIA for SARS-CoV-2 IgM and IgG antibodies. Here, we detected and quantified the results of samples containing different concentrations of SARS-CoV-2 IgM and IgG  using the two methods mentioned above. In comparison with the PDA-based LFIA, the AuNP-based commercial LFIA could barely detect low concentrations of COVID-19 antibodies, as shown in To make it convenient for users to easily perform the quantification, we developed software with the functions of test line extraction and concentration calculation. After taking a photo of the three-line strip with the reader a, users In conclusion, we successfully developed a PDA-based lateral flow immunoassay technique for the accurate and large-scale screening of COVID-19. Compare to other LFIA methods for COVID-19 diagnosis , two inn


Omega-3 polyunsaturated fatty acids (n-3 PUFAs), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been increasingly studied due to their beneficial health effects. N-3 PUFAs are particularly abundant in the brain and retina, where they play various roles that are important to the maintenance of normal function in those organs. The present study aimed to evaluate the FA profile of lamb brain and retinal tissues after they were fed three experimental diets supplemented with an EPA-rich microalga for 21 days. The microalga was delivered in a different format in each one of the diets ; therefore, its efficiency in altering the FA profile of brain and retina was evaluated for each diet. Overall, our results demonstrated that the brain EPA content remained unchanged after EPA supplementation, in contrast with the retinal EPA, which was very responsive to microalga supplementation.Nannochloropsis sp. microalga. Their brains and retina were collected for FA characterization. Overall, the brain FA profile remained unchanged, with little alteration in omega-3 docosapentaenoic acid (DPA) enhancement in both the hippocampus and prefrontal cortex. Retinal tissues were particularly responsive to the dietary intervention, with a 4.5-fold enhancement of EPA in the freeze-dried-fed lambs compared with the control lambs. We conclude that retinal tissues are sensitive to short-term n-3 PUFA supplementation in lambs.Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have special physiological functions in both brain and retinal tissues that are related to the modulation of inflammatory processes and direct effects on neuronal membrane fluidity, impacting mental and visual health. Among them, the long-chain (LC) n-3 PUFAs, as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are of special importance. Scarce data are available about the fatty acid (FA) composition of the ruminant brain in response to dietary intervention. However, we decided to examine the brain and retina FA composition of lambs supplemented with an EPA-rich microalga feed for 21 days, as it is known that despite the extensive biohydrogenation of dietary PUFAs in the rumen, ruminants can selectively accumulate some n-3 LC-PUFAs in their brain and retinal tissues. Twenty-eight male lambs were fed a control diet, or the same diet further supplemented with  Fatty acids (FAs) are the most abundant organic compounds in the brain. More than 90% of polyunsaturated FAs (PUFAs) in the mammalian brain are composed of long-chain PUFAs  such as arachidonic acid AA, 20:4n-6) and docosahexaenoic acid  . Most ofn-6 and dIn addition to the brain, retinal tissue also is rich in lipids, which comprise approximately 20% of the retina’s dry weight. Retinal membrane PLs have the highest level of LC-PUFAs of any tissue in humans (approximately 33%) ,7,8. TheThe brain lipids of ruminant species, especially cattle, have been well characterized and appear to be very similar to those of the human brain in terms of both content and composition . AlthougThe dietary supplementation of microalgae has been shown to enhance n-3 LC-PUFAs along lambs’ gastrointestinal tracts . Moreove2) with ad libitum access to clean water. The lambs were sorted into four experimental groups with seven replicates per group. The experimental diets included a control diet (C diet), consisting of pellets containing dehydrated lucerne, barley and soybean meal and no added sources of EPA, and three diets supplemented with the microalga Nannochloropsis sp., designed to provide approximately 3 g of EPA per kg of diet dry matter (DM). The average content of EPA (mg EPA/g product) in each microalgal format was 235 in the Nannochloropsis oil, 22.7 in the spray-dried Nannochloropsis oceanica and 30.8 in the lyophilized Nannochloropsis oceanica. Nannochloropsis sp.-containing diets were composed of the C diet plus 123 g/kg of spray-dried Nannochloropsis oceanica biomass (SD diet); 92 g/kg freeze-dried Nannochloropsis oceanica biomass (FD diet); and 12 g/kg of Nannochloropsis sp. free-oil (O diet)  . The triAfter the end of the third week, the animals were slaughtered using a captive bolt. This was followed by exsanguination. The brain tissue was removed whole. It retained its shape and landmarks in spite of the captive bolt damage. The brain was cut on a sagittal plane and divided into two hemispheres, which were then frozen at −80 °C. After thawing, the different parts were individualized from the right hemisphere , stored tapetum lucidum (RTL) were individualised  and dimethyl acetals (DMAs) of the brain, RTL tissues and liver samples were prepared by acid-catalysed transesterification in methanol . In plasFatty acid methyl esters and DMAs were analysed by gas chromatography with flame ionization detection (GC-FID), using a Shimadzu GC 2010-Plus  equipped with an SP-2560  capillary column. The injector and detector temperatures were maintained at 220 °C and 250 °C, respectively. The carrier gas was helium at a constant flow of 1 mL/min. The GC oven temperature began at 50 °C for 1 min, then increased to 150 °C at 50 °C/min, held for 20 min, increased to 190 °C at 1 °C/min, and finally increased to 220 °C at 2 °C/min and held for 40 min. The identification of FAMEs was achieved by a comparison of the fatty acid retention times with those of commercial standards  and with published chromatograms ,19. Addip  <  0.05. The TFA + DMA content is presented in mg/g DM, and the FA individual composition is presented in % of TFA + DMA (g FA/100 g TFA + DMA). The sparse partial least squares discriminant analysis (sPLSDA) was performed using MetaboAnalyst 5.0 software, using the centred log ratio transformed FA data as input. FAME and DMA data were analysed as a completely randomised experimental design using the MIXED procedure of SAS 9.4 . Diet was used as a fixed factor, and the animal was used as the experimental unit. Feed intake data were analysed as a completely randomised block design, in which an individual lamb was used as the experimental unit and the model included the treatment and initial live weight block as the fixed factors. The least square means and standard error of the mean (SEM) were reported, and the main effects and their interactions were considered significant at p > 0.05) [The feed intake averaged 1.19 ± 0.13 kg (mean ± standard error of the mean) of DM/day during the experiment. It did not differ among treatments ( > 0.05) . A briefNannochloropsis-diet-supplemented lambs (p < 0.05). The FA 14:0, 20:4n-6 and 20:5n-3 were only present in the Nannochloropsis-supplemented diets; therefore, their feed intake was zero in the control-fed lambs. The total FA intake differed between control and Nannochloropsis-supplemented lambs (p < 0.05), being higher in the latter.Apart from 16:0, the intake of all the FAs analysed differed between the control and The total FA and DMA (TFA + DMA) content (mg/g DM) and composition (g/100 TFA + DMA) of grey and white matter are presented in p > 0.05) were observed for any of the FAs and DMAs in both the grey and white matter.No major effects of microalgal supplementation (p < 0.05) lower in the C and SD diets and higher in the O and FD diets. In the prefrontal cortex, this FA was higher in all Nannochloropsis-supplemented lambs compared to C-fed lambs. Regarding 22:5n-6: it was significantly higher in the hippocampus in the C-fed lambs when compared to the Nannochloropsis-supplemented lambs. In the prefrontal cortex, this FA tended (p = 0.056) to follow a similar pattern to what was found in the hippocampus.Significant differences in the FA composition were observed in the hippocampus and prefrontal cortex . In the p < 0.05) in the hippocampi and prefrontal cortices of C lambs, and its highest values were found in the tissues of lambs fed with the FD supplement. DPA was lower (p < 0.05) in these tissues in O-fed lambs than in SD-fed lambs.DPA had its lowest values (p > 0.05) the EPA or DHA concentrations in the hippocampus or prefrontal cortex. The sum of EPA + DHA averaged 10% in the hippocampus and 11% in the prefrontal cortex. The total PUFA and n-3 PUFA contents averaged 22% and 12% in both the hippocampus and prefrontal cortex, respectively. Supplement treatments did not affect (p > 0.05) by dietary treatment. The content of TFA + DMA was not affected  in SD lambs than in all other treatments. Regarding 20:3n-9, a higher content was found in both the C- and SD-fed lambs when compared to the remaining groups. The content of 20:3n-6 was lower in C-fed lambs when compared to the remaining treatments, and 22:4n-6 was higher in both C- and SD-fed lambs when compared to the remaining treatments. Both EPA and DPA were higher in the microalgae-biomass-fed lambs, although there were no differences between the SD- and FD-fed lambs. When compared to the C treatment, biomass-fed lambs had 4.6 times more EPA and twice more DPA in their RTL tissues.In RTL tissues, the p > 0.05).The DHA content in RTL tissues did not differ among treatments (p < 0.001). The sum of the EPA + DHA averaged 7% TFA. N-3 PUFA averaged 10% ± 1.0, corresponding to approximately 45% of the total PUFAs.In the partial sums evaluated, the AA/EPA ratio differed between treatments, being higher in the C-fed lambs when compared to the remaining treatments , DHA (≈9%) and AA (≈5% of TFA + DMA). A similar pattern was also observed in the bovine brain regions [c9-18:1 (≈24%), AA (≈6%) and DHA . It was shown that linoleic acid (LA), often the major PUFA in muscle, and α-linolenic acid (ALA) were only present at very low levels. In fact, the longissimus lumborum muscles of the same animals presented LA and ALA proportions of circa 9 and 1% [The most abundant FAs found in the ovine brain were 18:0 (≈18%), 16:0 (≈17%),  regions , with th9 and 1% , contras9 and 1% . Moreove9 and 1% .Nannochloropsis oceanica-supplemented diets appeared to produce a higher n-3 LC-PUFA enhancement in the lambs’ brains when compared to O and SD because freeze-drying better protects the integrity of the microalgal cell wall, reducing the access of ruminal microbiota to the n-3 LC-PUFA inside the cell. In ruminants, almost 90% of dietary lipids reach the duodenum as non-esterified saturated FAs [Nannochloropsis-supplemented treatments [Nannochloropsis-supplemented treatments  have been attributed to DHA and EPA ,35. HowePlasmalogens are a subclass of glycerophospholipids that comprise part of biological membranes, including the plasma membrane and the membranes of intracellular organelles, affecting their biophysical properties. They are quantitively important in membranes of neuronal tissues, including the brain and the retina, and are associated with neurological and psychiatric disorders or are involved in the regulation of retinal vascular development, respectively ,37. In ttapetum [tapetum lucidum is a biologic reflector system that is commonly present in the eyes of vertebrates. It enhances visual sensitivity at low light levels by providing light-sensitive retinal cells with a second opportunity for photon–photoreceptor stimulation. Ovine tapetum lucidum belongs to the choroidal fibrous type, and the reflective material is made of collagen [tapetum [tapetum tissue. The retina is a thin, highly organised neural tissue lining the posterior aspect of the eye. It is responsible for initiating vision by transducing light into neural signals . The vistapetum . Therefocollagen  that con[tapetum . When coc9-18:1 averaged 26% and DHA averaged 6.4%. AA averaged 6%, in line with it being the most abundant omega-6 PUFA in the retina [Studies with rodents demonstrated that the FA composition of the retina is influenced by diet ,49,50,51e retina ,8. Contrary to what was observed in the brain, EPA supplementation increased the EPA content in the RTL tissues. In the C lambs, EPA averaged 0.18% TFA + DMA, which was in line with human EPA retinal content . The EPANannochloropsis-supplemented diets in the sPLSDA analysis. This shows, once again, that the RTL tissues seem to have been much more sensitive to dietary intervention. The high responsiveness of the retina is evident in rodent studies, in which EPA contents of 6 to 35 times greater have been reported following EPA supplementation [The 4.6-fold increase was achieved despite the short duration of EPA supplementation. Consistent with a better responsiveness of RTL tissues to the experimental diets, control lambs were clearly separated from the lambs consuming entation ,55.As in the brain, no alterations in DHA content were observed between different treatments. The content of DHA in RTL tissues averaged 6.4% of the total TFA + DMA. This is considerably lower that what was reported in previous studies for ruminants in which the DHA content averaged approximately 20–30% ,56.Nannochloropsis sp. microalga, it was possible to conclude that the brain content of EPA was not responsive to dietary supplementation. However, the EPA content in the retina was highly responsive in lambs supplemented with Nannochloropsis, especially lambs consuming SD and FD diets. Although we could not confirm an advantage in freeze-drying over spray-drying Nannochloropsis oceanica with respect to the efficiency of EPA enhancement in the lambs’ retinal tissues, we can confirm their advantage over the free oil. Overall, our results suggest that RTL is a good target to evaluate the differences in n-3 LC-PUFA absorption due to the changes in rumen biohydrogenation associated with dietary interventions.After a short-term trail of EPA supplementation in lambs, achieved through feeding using three different diets containing


To develop powders constituted by such SLNs for nasal administration, herein, two different agents, namely sucrose and methyl-β-cyclodextrin (Me-β-CD), were evaluated as cryoprotectants. (2) Methods: SLNs were prepared following the melt homogenization method, and their physicochemical features were investigated by Raman spectroscopy, Scanning Electron Microscopy (SEM), atomic force microscopy (AFM) and X-ray Photoelectron Spectroscopy (XPS). (3) Results: SLN size and zeta potential values changed according to the type of cryoprotectant and the morphological features investigated by SEM showed that the SLN samples after lyophilization appear as folded sheets with rough surfaces. On the other hand, the AFM visualization of the SLNs showed that their morphology consists of round-shaped particles before and after freeze-drying. XPS showed that when sucrose or Me-β-CD were not detected on the surface , then a DA surfacing was observed. In vitro release studies in Simulated Nasal Fluid evidenced that DA release, but not the GSE one, occurred from all the cryoprotected formulations. Finally, sucrose increased the physical stability of SLNs better than Me-β-CD, whereas RPMI 2650 cell viability was unaffected by SLN-sucrose and slightly reduced by SLN-Me-β-CD. (4) Conclusions: Sucrose can be considered a promising excipient, eliciting cryoprotection of the investigated SLNs, leading to a powder nasal pharmaceutical dosage form suitable to be handled by PD patients.(1) Background: DA-Gelucire Undoubtedly, in the field of neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease (PD), several issues in overcoming the Blood–Brain Barrier (BBB) are to be faced. The protection role exerted by the BBB mainly consists of limiting the access of xenobiotics into the brain compartment and, consequently, low bioavailability and limited brain penetration of administered drugs occurs. It prompts us to find alternative strategies to reach the Central Nervous System (CNS) in a comfortable way for patients affected by neurological disorders.Currently, for patients affected by neurological disorders, a very promising approach is constituted by intranasal drug administration since the olfactory mucosa is the region where the BBB is interrupted. The unique characteristics of the nasal mucosa enable it to bypass the BBB, while its CNS connections allow drug transport directly into the brain ,2,3. In ® 50/13, a self-emulsifying lipid, as a component of SLNs, is capable of increasing the drug-loading of hydrophilic active principles . Th. Th−1, sn chains . In the brations ,23. In todextrin ,25.−1, 1000–1200 cm−1, 1230–1350 cm−1 and 2800–3000 cm−1, leading to a wider and more flattened shape, probably due to the presence of the SNF.When Raman spectra were acquired after SLN interactions with SNF b, the prSEM observations were carried out for DA-co-GSE-SLNs formulations selected as representative. SEM microphotographs of DA-co-GSE-SLNs and DA-co-GSE-SLNs-sucrose (both before and after freeze-drying using sucrose as a cryoprotectant) are shown in Along with SEM observations, we have also performed morphological characterization of DA-co-GSE-SLNs and GSE-ads-DA-SLNs by using AFM before freeze-drying , providiAfter freeze-drying , we obseXPS analysis of the developed formulations was carried out in order to gain information on the surface presence of the neurotransmitter and—in the cases of the cryoprotected SLNs—of the cryoprotectant. The surface atomic composition of all the formulations, with or without sucrose (or Me-β-CD), was reported in To evaluate the cyto-compatibility of the pure substances DA and GSE as well as of DA-co-GSE-SLNs and GSE-ads-DA-SLNs (these last lyophilized with and without cryoprotectant agents) with RPMI 2650 cells , the viability of the mentioned cells when treated with such substances was studied. In The aim of the present work was to evaluate the effect of a small molecule  and of an oligomer  cryoprotectant on the lyophilization of a suspension of GSE/DA-SLNs. It is in order to deliver the resulting solid nanocarriers as powders by intranasal route as a new approach for PD treatment. As shown in ®. Such interactions do not occur when DA and GSE are co-encapsulated in SLNs with cryoprotectants. Furthermore, the same release profile observed for DA-co-GSE-SLNs-Me-β-CD and DA-co-GSE-SLNs-sucrose is not surprising since the same amount of GSE is employed to prepare both these PEGylated SLNs.To justify the results of the in vitro release kinetic study of DA and GSE shown in 2890/I2850 ratio is an indication of chain packing and conformational disorder, while the I1115/I1050 cm−1 ratio refers to the fluidity within the hydrocarbon chains [−1, 1000–1200 cm−1, 1230–1350 cm−1and 2800–3000 cm−1, leading to a flattened shape, probably due to the presence of the SNF. Thus, in perspective, Raman spectroscopy confirms its interesting potential when applied to provide more detailed information on these systems.As for the investigation aimed to evidence possible interactions with nasal fluids of cryoprotected SLNs, we have recently reported that a useful approach is based on the use of Raman spectroscopy , herein n chains . In the ® can displace DA from the external surface of the SLNs, thus determining DA leakage. Overall, such a reduction in DA levels did not cause the absence of the neurotransmitter delivered in SNF, as shown in On the other hand, XPS provided important information on the surface presence of the neurotransmitter as well as even the cryoprotectant when it occurs. Indeed, XPS analysis showed in DA-co-GSE-SLNs-sucrose after freeze-drying a negligible sucrose presence and detection of DA on the surface . A similIn the development of intranasal drug delivery systems for brain targeting, it is necessary to assess its cytotoxicity through the nasal epithelium. Concerning the biological evaluation of the investigated SLNs, the RPMI 2650 cell line was herein adopted as they represent a cell model widely explored for mimicking human nasal epithelium ,31,32,33Regarding the effect of the GSE effect on the cells, we observed a statistically significant decrease in RPMI 2650 viability at concentrations higher than 25 µg/mL at any incubation time point , confirm® 50/13 was kindly donated by Gattefossé . Dopamine hydrochloride, sucrose, carboxyl ester hydrolase , Tween® 85, as well as the salts used for buffer preparation, were bought from Sigma-Aldrich . Methyl-β-cyclodextrin  was a gift from Wacker Chemie  and kept in a desiccator until use. For cell cultures, advanced Minimum Essential medium (A-MEM) was purchased from Gibco-Thermo Fisher Scientific . Advanced Minimum Essential medium (A-MEM) was purchased from Gibco-Thermo Fisher Scientific . Heat-inactivated fetal bovine serum (FBS) was purchased from Euroclone S.p.A . GutaMAX™ Supplement was acquired from Biowest . Trypsin EDTA 0.25% was purchased from Elabscience . Alamar Blue and Resazurin were purchased from Bio-Rad  and Biotium , respectively. In this work, double distilled water was used, and all other chemicals were of reagent grade.Grape seed extract containing ≥ 95.0% proanthocyanidins was received as a gift by Farmalabor . Gelucire® 50/13 SLNs were prepared following the melt homogenization method. Briefly, Gelucire® 50/13 (60 mg) was melted at 70 °C. GSE (6 mg) were dispersed in the aqueous phase made of surfactant  and 1.37 mL diluted acetic acid  in a separate vial under homogenization at 12,300 rpm with an Ultra-Turrax model T25 apparatus  and left to equilibrate for 30 min at 70 °C. Then, 10 mg of DA was added to the aqueous phase, the resulting mixture was emulsified with the melted Gelucire® 50/13, and the emulsion was homogenized at 12,300 rpm for 2 min by Ultra-Turrax system. Then, the nanosuspension was cooled at room temperature and allowed to achieve DA co-encapsulating GSE SLNs. Such SLNs were centrifuged , the pellet was harvested and re-suspended in distilled water for further studies, and the supernatant was discarded. Throughout the manuscript, the resulting SLNs were abbreviated “DA-co-GSE-SLNs”. For GSE-adsorbing DA SLNs, DA-loaded SLNs were first formulated as reported elsewhere [g for 45 min (Eppendorf 5415D), and the pellet was re-dispersed in distilled water, whereas the supernatant was discarded. Throughout the manuscript, the resulting SLNs were abbreviated as “GSE-ads-DA-SLNs”.DA-loaded Gelucirep = 0.1 mbar; Lio Pascal 5 P, Milan, Italy). Below, the resulting SLNs were abbreviated “DA-co-GSE-SLNs-sucrose (or -Me-β-CD)” and “GSE-ads-DA-SLNs-sucrose (or -Me-β-CD)”.For both DA-co-GSE-SLNs and GSE-ads-DA-SLN, the effect of two cryoprotectant excipients was studied following hints in the literature . After p3OH 70:30 (v:v)) was adopted as mobile phase. The isocratic mode was selected for column elution at the flow rate of 0.7 mL/min, and, under such chromatographic conditions, the retention times of DA and GSE were found to be equal to 5.5 min and 12 min , and the obtained supernatant was subjected to HPLC analysis, as above. The encapsulation efficiency (E.E.%) was calculated by Equation (1):To determine DA and GSE content, herein, quantification of the active principles occurred after freeze-drying of the different types of cryoprotected SLNs, which underwent enzymatic digestion by esterases . The enzFurthermore, drug-loading (D.L.) was also calculated by Equation (2):This study was performed in triplicate.v/v) prior to being analyzed for particle size. For ζ-potential measurements, laser Doppler anemometry technique was adopted  by using the same dilution as described above for size analysis.Mean particle size and polydispersity index (PDI) of the SLNs in the presence and in the absence of cryoprotectant were determined according to Photon Correlation Spectroscopy (PCS) of the ZetasizerNanZS  apparatus. Samples of SLNs were analyzed in terms of particle size, PDI and zeta potential before and after freeze-drying. They were introduced in folded capillary zeta cells with a measurement angle of 173° Backscatter. Particle size and PDI values of the SLNs were measured at 25 °C by re-dispersing each of them in 1 mL of double distilled water, followed by a brief sonication. Then, the nanosuspensions underwent a further dilution in double distilled water , and the amounts of the neurotransmitter and the antioxidant GSE delivered were quantified in the resulting supernatants by HPLC, as described above, and plotted against the time. All the release experiments in SNF were carried out in triplicate.The DA and GSE release in SNF  /mm grating. The parameters used for Raman spectra acquisitions were 2 s exposure, 20 accumulations, 100% of laser power and 60–300 s bleaching. In order to gain insight into the interactions occurring between SLNs (provided or not of cryoprotectant) and the nasal compartment, freeze-dried SLNs were incubated in 20 mL of SNF at 37 °C for 3 h in an ISCO thermostated incubator  without any agitation. Once the incubation was over, a few drops of the resulting suspensions were placed on different slides and allowed to evaporate overnight prior to being subjected to Raman investigations.After freeze-drying SLNs in the presence and in the absence of cryoprotectants, Raman spectra were recorded using an InVia Renishaw microscope  equipped with a 532 nm and 633 nm laser in the presence of a 100× objective. The specimens were placed onto a microscope slide, and the range examined was 400–3500 cmEnergy-dispersive X-ray analysis (EDX) and Scanning Electron Microscopy (SEM) were performed using a VegaII microscope  with a Quantax elemental detector . The specimens were placed onto a microscope stub, and a sputter coater  was used to cover specimens with a thin gold layer. Then, the specimens were introduced into the SEM chamber and analyses were performed using a high voltage of 20 kV.v/v) with ultrapure water and pipetted onto the silicon wafers. Samples were then incubated for 30 min to allow adherence of particles to the wafer’s surface. Excess water was shaken off, and samples were mounted onto the stage of a NanoWizard®-3 NanoScience AFM , which was vibration-damped and located in an acoustic isolation chamber.Freshly cut silicon wafers (0.5 cm × 0.5 cm) were glued onto a microscopic glass slide and thoroughly cleaned with pure isopropanol and ultrapure water. Afterward, SLN suspensions were diluted 1:10 . For curve-fitting of the high-resolution spectra, the commercial MultiPak software version 9.9.0.8 was used. Adventitious carbon C1s was set as the reference charge (284.8 eV).XPS analyses were performed using a scanning microprobe PHI 5000 VersaProbe II purchased from Physical Electronics . The instrument was equipped with a micro-focused monochromatized AlKα X-ray radiation source. Freeze-dried SLNs (with or without cryoprotectant) were examined in HP mode with an X-ray take-off angle of 45°, with an instrument base pressure of ~102 polystyrene tissue culture flasks using A-MEM. The medium was supplemented with 4 mM GlutaMAX™ and 2.5% FBS. The cell cultures were maintained at 37 °C in a >95% humidified atmosphere of 5% CO2 in air, with media changes on alternative days [The RPMI 2650 cells, sourced from the European Collection of Cell Cultures, Health Protection Agency, were donated by Dr. Katja Kristan . They were cultured in 25 cmive days ,30.® Omega multi-plate reader  at 560 nm (excitation) and 590 nm (emission). Cryoprotected SLNs based on the concentration of DA were diluted using A-MEM in order to obtain the following DA concentrations: 100, 75, 50 and 25 µM. Once DA concentration was fixed in the SLNs, then the GSE range of concentrations was found to be between 23.8 µg/mL and 0.02 ng/mL. Moreover, for SLNs cryoprotected by sucrose and Me-β-CD, 0.8–0.1 mg/mL and from 1 mg/mL to 0.078 mg/mL were the ranges examined for each excipient, respectively. Each formulation was tested two to three times in triplicate.Resazurin protocol  was used to assess RPMI 2650 cell viability . For thep < 0.05. Biological data are presented as means ± standard error (s.e.m.). The effect of treatments was analyzed using GraphPad Prism 9.0.0 software for Windows  by ANOVA. The Dunnett post hoc test and the Sidak multiple comparison test were performed. Particularly, the Dunnett test compared the means of several experimental groups with the mean of a control group to determine whether there was a difference; the Sidak test was used to compare viability values regarding the same concentration of samples with and without cryoprotectants. Differences were considered statistically significant at p < 0.05.For physicochemical data, mean ± SD and statistical evaluation were obtained by Prism v. 5.0 . Multiple comparisons were based on one-way analysis of variance (ANOVA), with Bonferroni’s post hoc test, and differences were considered significant when Herein, SLNs loaded with two hydrophilic active principles, namely DA and GSE, were formulated in the presence of sucrose or Me-β-CD as cryoprotectant agents in view of the development of a nasal solid dosage form for PD application. Particle size retention at 4 °C together with RPMI 2650 cell viability evaluation evidenced some benefits to adopt sucrose better than Me-β-CD. Thus, the physical stability study evidenced the positive role played by sucrose as a cryoprotectant, which considerably affects the stability of the SLNs studied, as proved by particle size retention up to three months of storage at 4 °C. Overall, considering the improvement of SLN stability, the whole freeze-drying cycle using sucrose as a cryoprotectant is advantageous in this case. In addition, it should also be taken into account that RPMI 2650 full viability was retained up to 24 h of incubation via the use of sucrose rather than Me-β-CD as a cryoprotectant of SLNs. Finally, the outcomes of this research work will constitute a platform for further studies addressed to obtain the aerosolization of the nasal powders herein described and build the device containing such pharmaceutical dosage forms.